Difference between revisions of "Team:Sherbrooke/Experiments"

Line 4: Line 4:
  
 
<h1>Experiments &amp; Protocols</h1>
 
<h1>Experiments &amp; Protocols</h1>
 +
 +
<a href="#Biology">Jump to biology section</a>
  
 
<h2>Projects modules</h2>
 
<h2>Projects modules</h2>
Line 671: Line 673:
 
<a href="#top_menu_under">Back to top</a>
 
<a href="#top_menu_under">Back to top</a>
  
 +
<span id="Biology"> &nbsp; </span>
 +
<h2>Biology section</h2>
 +
<ul>
 +
<li><a href="#Bio_Exp">Experiments section</a></li>
 +
<li><a href="#Bio_Protocol">Biology Protocols</a></li>
 +
</ul>
 +
<span id="Bio_Exp"> &nbsp; </span>
 +
<h2>Biology Experiments</h2>
 +
<ul>
 +
<li><a href="#Recom_construct">Recombineering cassette construction</a></li>
 +
<li><a href="#Recom_test">Recombineering cassette test</a></li>
 +
<li><a href="#Cyclic_del">Cyclic deletion casette test</a></li>
 +
<li><a href="#Biobrick_transfert">Transfert in Biobrick standardised system</a></li>
 +
</ul>
 +
<span id="Recom_construct"> &nbsp; </span>
 +
<h3>Recombineering cassette construction</h3>
 +
<p style="text-align:justify">
 +
In order to test our BIOBOT platform (which could ultimately do automated MAGE experiment), a recombineering experiment has to be set-up. While a lot of reliable selectable markers are known, we can’t say the same about counter selectable ones. Many counter selectable markers for recombineering are actually toxins isolated from conjugative plasmids. pVCR94 is a plasmid isolated during the 1994 cholerae outbreak in a Rwanda refugee camp. This conjugative plasmid carries resistance to a lot of antibiotics and its regulation was recently investigated by
 +
<a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207636/> Carraro et al. 2015</a>.
 +
</p>
 +
<p style="text-align:justify">
 +
Like most conjugative plasmids, it possesses many stabilisation and partition systems. One of which is a powerful toxin-anti-toxin system. The toxin was firstly tested for its ability to kill cells compared to other known toxins. To do so, mosT, ccdB, mazF and pVCR94’s toxin: vcrx028 were cloned in pBAD30 to obtain arabinose triggered killswitches. To do so, primers specified in the design sections were used on different template: pSXT, ptac-ccdb-casette, gBlock_FG1, pVCR94. See design section here:
 +
<a href=https://2015.igem.org/Team:Sherbrooke/Design#Toxins> Arabinose inducible toxin</a>.
 +
</p>
 +
 +
<span id="Recom_test"> &nbsp; </span>
 +
<h3>Recombineering cassette test</h3>
 +
<p style="text-align:justify">
 +
The different killswitches (pBAD30-[mosT;ccdB;mazF;vcrx028]) were tested (see <a href=https://2015.igem.org/Team:Sherbrooke/Experiments#toxins_test>here</a> for the protocol) to investigate if they worked and to know which one was the best. We found that mosT and vcrx028 were the best counter-selectable markers (see <a href=https://2015.igem.org/Team:Sherbrooke/Results#toxins_test>here</a> for the results)
 +
</p>
 +
<p style="text-align:justify">
 +
Our multi-copy plasmid results shown that mosT and vcrx028 were the best candidates for our system, but we needed to test them in single-integrated-copy for it to be more representative. Therefore, we amplified cassettes containing all the needed elements of the killswitches from pBAD30-[mosT;vcrx028] with primers adding homologies that allow lambda-red mediated recombination in the lacZ truncated gene of Escherichia coli BW25113. After confirmation of the recombinants, they were tested and it shown that both killswitch were still working even in single copy. The main difference is that after 10-12 hours, some mutants that inactivated the integrated killswitch began growing. All the tested clones for mosT shown that behavior, but only 1 of the 3 vcrx028 clones did. <a href=https://2015.igem.org/Team:Sherbrooke/Experiments#toxins_test>See the protocol</a> or <a href=https://2015.igem.org/Team:Sherbrooke/Results#toxins_integrated_test>See the results</a>
 +
</p>
 +
 +
<span id="Cyclic_del"> &nbsp; </span>
 +
<h3>Cyclic deletion system test</h3>
 +
 +
<span id="Biobrick_transfert"> &nbsp; </span>
 +
<h3>Transfert in Biobrick standardised system</h3>
 +
 +
<span id="Bio_Protocol"> &nbsp; </span>
 +
<h2>Biology Protocols</h2>
 +
<ul>
 +
<li><a href="#toxins_test">Recombineering cassette test</a></li>
 +
<li><a href="#Medium">Medium and reagent use</a></li>
 +
<li><a href="#PCR_protocol">PCR general protocol</a></li>
 +
<li><a href="#Chem_transfo">Chemical transformation</a></li>
 +
<li><a href="#Electro_transfo">Electroporation</a></li>
 +
<li><a href="#Recombineering">Recombineering</a></li>
 +
<li><a href="#miniprep">DNA miniprep</a></li>
 +
<li><a href="#restriction_digest">DNA digestion with restriction enzymes</a></li>
 +
<li><a href="#Gibson">Gison assembly</a></li>
 +
 +
</ul>
 +
 +
<span id="toxins_test"> &nbsp; </span>
 +
<h3>Recombineering cassette test</h3>
 +
<p style="text-align:justify">
 +
To test the effect of the toxins on the cells containing the different killswiches:
 +
</p>
 +
<ul>
 +
<li>2 ul of culture was used to inoculate 198 ul of LB-ampicillin-Glucose 5% or LB-ampicillin-arabinose 1%</li>
 +
<li>That was done in a 96-wells plate put at 30°C</li>
 +
<li>The OD[600] was measured at different intervals during 16-24 hours using a plate reader</li>
 +
</ul>
 +
 +
<a href="#Biology">Back to Biology Menu</a>
 +
 +
<span id="Medium"> &nbsp; </span>
 +
<h3>Medium and reagent use</h3>
 +
<h4>Medium</h4>
 +
<p style="text-align:justify">
 +
The only medium used in this project was LB in broth and agar form. Both comes from BIOBASIC, their catalog numbers are SD7002(S518) and SD7003(S519).
 +
</p>
 +
<h4>Antibiotics</h4>
 +
<p style="text-align:justify">
 +
Here are the complete list of antibiotics used in this project with the working concentration for each:
 +
</p>
 +
<ul>
 +
  <li>Ampicilin: 100 µg/mL</li>
 +
  <li>Chloramphenicol: 34 µg/mL</li>
 +
  <li>Kanmycin: 50 µg/mL</li>
 +
</ul>
 +
<h4>Other Regeants</h4>
 +
<ul>
 +
  <li>D-Glucose: used at 5% w/v</li>
 +
  <li>L-Arabinose: used at 1% w/v</li>
 +
  <li>Molecular Grade Water</li>
 +
  <li>TFBI:30 mM KOAc, 100 mM RbCl, 10 mM CaCl2, 50 mM MnCl2, 15 % glycerol, pH 5.8 (adjusted with acetic acid), 0.22 µm filtered.</li>
 +
  <li>TFBII:10 mM MOPS or PIPES, 75 mM CaCl2, 10 mM RbCl, 15 % glycerol, pH 6.5 (adjust with KOH), 0.22 µm filtered.</li>
 +
</ul>
 +
<h4>Strains</h4>
 +
<ul>
 +
  <li><i>Escherichia coli K-12 substrain EC100</i></li>
 +
  <li><i>Escherichia coli K-12 substrain BW25113</i></li>
 +
</ul>
 +
 +
<a href="#Biology">Back to Biology Menu</a>
 +
<span id="PCR_protocol"> &nbsp; </span>
 +
<h3>PCR general protocol</h3>
 +
<p style="text-align:justify">
 +
The polymerase used for all experiment was Veraseq from Enzymatics<sub>™</sub>. This polymerase adds 2 kb of DNA per minute. PCR were always done following this fact. All PCR except those specified in the text were made with 50 Celsius degrees as annealing temperature. The recipe for all PCR mix is as follow:
 +
</p>
 +
<table id=”t01”>
 +
<tr>
 +
  <th>Component</th>
 +
  <th>Volume for 1 Reaction</th>
 +
</tr>
 +
<tr>
 +
  <td>Molecular Grade Water</td>
 +
  <td>17.5 µL</td>
 +
</tr>
 +
<tr>
 +
  <td>5X Veraseq Buffer</td>
 +
  <td>5 µL</td>
 +
</tr>
 +
<tr>
 +
  <td>10 mM dNTP</td>
 +
  <td>0.5 µL</td>
 +
</tr>
 +
<tr>
 +
  <td>Forward Primer</td>
 +
  <td>0,5 µL</td>
 +
</tr>
 +
<tr>
 +
  <td>Reverse Primer</td>
 +
  <td>0.5 µL</td>
 +
</tr>
 +
<tr>
 +
  <td>Template DNA (1 ng/µL)</td>
 +
  <td>1 µL</td>
 +
</tr>
 +
<tr>
 +
  <td>Veraseq Polymerase</td>
 +
  <td>0.25 µL</td>
 +
</tr>
 +
</table>
 +
<p style="text-align:justify">
 +
The PCR mixes were all done on ice and we followed manufacturer’s recommendation for denaturation and elongation temperature.
 +
</p>
 +
 +
<a href="#Biology">Back to Biology Menu</a>
 +
 +
<span id="Chem_transfo"> &nbsp; </span>
 +
<h3>Chemical transformation</h3>
 +
<p style="text-align:justify">
 +
For TFBI and TFBII composition, please refer to other reagents section. Sometimes, chemical transformation can be an advantage compared to electroporation. Basically, the DNA source of the transformation doesn’t need to be pure for a chemical transformation and can be highly concentrated in salt. You can put as much a 10% of the total volume in DNA in each transformation. The cells can be frozen at -80 Celsius degrees and thawed when needed. Here is the total procedure used in our lab to prepare chemically competent cells:
 +
</p>
 +
<ul>
 +
  <li>Inoculate a single colony into 5 ml LB broth.</li>
 +
  <li>Incubate 37°C O/N with 200 rpm agitation.</li>
 +
  <li>Subculture the O/N 1:100 in LB + 6 mM MgSO4 (typically 250 ml).</li>
 +
  <li>Grow to OD600 = 0,48 (0,4-0,6 is good, should take 2-3h).</li>
 +
  <li>Centrifuge 6,000 rpm 5 min at 4°C.</li>
 +
  <li>Gently resuspend pellet in 1/2,5 volume unit ice cold TFBI. (for 250 ml, use 100 ml TFBI ; 50 ml/bottle.</li>
 +
  <li>. Combine the resuspended cells in one bottle. Keep all steps on ice. Incubate on ice for 5 min.</li>
 +
  <li>Centrifuge 5,000 rpm 5 min 4°C.</li>
 +
  <li>Resuspend pellet in 1/25 original volume ice cold TFBII (for 250 ml original, use 10 ml TFBII).</li>
 +
  <li>Incubate on ice 15-60 min. Put 100 ul per tube and flash freeze tubes with liquid nitrogen. Store at -80°C.</li>
 +
  <li>When needed, cells can be thawed 15 minutes on ice before using them for transformation</li>
 +
  <li>Add as much as 10 µL of DNA to a cell aliquot</li>
 +
  <li>Incubated 45 seconds at 42 Celsius degrees then add 1 mL of cold LB</li>
 +
  <li>Let cells recuperate 1 hour at 37 degrees before plating them on the right selective plate.</li>
 +
</ul>
 +
 +
<a href="#Biology">Back to Biology Menu</a>
 +
 +
<span id="Electro_transfo"> &nbsp; </span>
 +
<h3>Electroporation</h3>
 +
<p style="text-align:justify">
 +
To execute this protocol, make sure you have access to an electroporater and be carefull handling the device, as its function is to give electric pulses.
 +
<ul>
 +
  <li>Start an overnight pre-culture of your cells to be electroporated at 30 Celsius degrees.</li>
 +
  <li>Once fully grown (after overnight incubation), inoculated a 4 mL LB broth per needed electroporation by diluting the pre-culture 1:50.</li>
 +
  <li>Incubate the cells at 30 Celsius egrees with 200 rpm agitation until it has an optical density (OD) of between 0.4 and 0.8 using a 1 cm optic cuvette at 600 nm in a spectrophotometer.</li>
 +
  <li>Then, transfers your culture in a falcon and incubated on ice for 15 minutes</li>
 +
  <li>Spin your cells at 7000 x g for 5 minutes.</li>
 +
  <li>Aspirate the supernatant and wash the pellet with 1 mL of cold sterile water.</li>
 +
  <li>Transfers the cells in a 1.5 mL microtube and centrifuge at 10 000 x g for 1 minute</li>
 +
  <li>Get rid of the supernatant and wash again with 1 mL of cold sterile water</li>
 +
  <li>Centrifuge at 10 000 x g for 1 minute</li>
 +
  <li>Get rid of the supernatant and wash again with 1 mL of cold sterile water</li>
 +
  <li>Centrifuge at 10 000 x g for 1 minute</li>
 +
  <li>Get rid of the supernatant and resuspend your cells in 40 µL of cold sterile water per needed electroporation (which brings them to 1000X concentration)</li>
 +
  <li>Dispatch 40 µL of cells in microtubes and add 1 µL of DNA 50 ng/µL (if possible).</li>
 +
  <li>Transfers the cells into 1 mm electroporation cuvettes.</li>
 +
  <li>Electroporate using 1,8 kV, 200 Ω, 25 µF and target 5.0 ms.</li>
 +
  <li>Resuspend cells in 1 mL of LB and let them recuperate 1 hour at 30 Celsius degrees (or 37 Celsius degrees).</li>
 +
  <li>Plate 200 µL of the transformation on a LB agar plate containing the right selection antibiotic.</li>
 +
<ul>
 +
 +
<a href="#Biology">Back to Biology Menu</a>
 +
 +
<span id="Recombineering"> &nbsp; </span>
 +
<h3>Recombineering</h3>
 +
<p style="text-align:justify">
 +
The recombineering technic ressembles a lot the electroporation one. There is two major adds and changes to the protocol. This comes from the fact that recombineering uses lambda red system. This system is (in our case) carried by a pSIM plasmid. Those plasmid are thermosensible (the origin of replication do not work at 37 degrees Celsius) and the lambda red system is heat induced. Those two fact explains the major changes in the protocol. The recombineering technique was developped by <a herf=”http://www.ncbi.nlm.nih.gov/pubmed/10829079”>Datsenko <i>et al.</i> (2000)</a> and uses three protein for the lambda phage: <i>bet, gam, exo</i>. <i>exo</i> firstly degrades one of the two strand of a double stranded DNA fragment through its 5’->3’ exonuclease activity. Then,<i> bet</i> will bind the single stranded DNA and insert it in the genome. <i> gam </i>, for instance, will inhibit endogenous nucleases.
 +
</p>
 +
<ul>
 +
  <li>Start an overnight pre-culture of your cells to be electroporated at 30 Celsius degrees.</li>
 +
  <li>Once fully grown (after overnight incubation), inoculated a 4 mL LB broth per needed electroporation by diluting the pre-culture 1:50.</li>
 +
  <li>Incubate the cells at 30 Celsius egrees with 200 rpm agitation until it has an optical density (OD) of between 0.4 and 0.8 using a 1 cm optic cuvette at 600 nm in a spectrophotometer.</li>
 +
  <li>Transfers the culture into a 42 Celisus degrees agitetive bath and incubate 15 minutes with 180 rpm agitation.</li>
 +
  <li>Then, transfers your culture in a falcon and incubated on ice for 15 minutes</li>
 +
  <li>Spin your cells at 7000 x g for 5 minutes.</li>
 +
  <li>Aspirate the supernatant and wash the pellet with 1 mL of cold sterilewater.</li>
 +
  <li>Transfers the cells in a 1.5 mL microtube and centrifuge at 10 000 x g for 1 minute</li>
 +
  <li>Get rid of the supernatant and wash again with 1 mL of cold sterile water</li>
 +
  <li>Centrifuge at 10 000 x g for 1 minute</li>
 +
  <li>Get rid of the supernatant and wash again with 1 mL of cold sterile water</li>
 +
  <li>Centrifuge at 10 000 x g for 1 minute</li>
 +
  <li>Get rid of the supernatant and resuspend your cells in 40 µL of cold sterile water per needed electroporation (which brings them to 1000X concentration)</li>
 +
  <li>Dispatch 40 µL of cells in microtubes and add 1 µL of DNA 50 ng/µL (if possible).</li>
 +
  <li>Transfers the cells into 1 mm electroporation cuvettes.</li>
 +
  <li>Electroporate using 1,8 kV, 200 Ω, 25 µF and target 5.0 ms.</li>
 +
  <li>Resuspend cells in 1 mL of LB and let them recuperate 1 hour at 30 Celsius degrees (or 37 Celsius degrees if you want to loose the pSIM plasmid).</li>
 +
  <li>Plate all of the transformation on a LB agar plate containing the right selection antibiotic (you can spin the cells and resuspend them in 100 µL to concentrate them).</li>
 +
<ul>
 +
 +
<a href="#Biology">Back to Biology Menu</a>
 +
 +
<span id="miniprep"> &nbsp; </span>
 +
<h3> DNA miniprep</h3>
 +
<p style="text-align:justify">
 +
The DNA minipreps were all done using the EZ-10 Spin Colomn Miniprep Kit<sub>™</sub>. Those are available commercially and distributed by Biobasic<sub>™</sub>. We have strictly followed manufacturer’s recomendation and obtained 50 µL of 50 ng/µL on average.
 +
</p>
 +
<a href="#Biology">Back to Biology Menu</a>
 +
<span id="restriction_digest"> &nbsp; </span>
 +
<h3>DNA digestion with restriction enzymes</h3>
 +
<p style="text-align:justify">
 +
It may be necessary in some cases to digest the vector in order to construct a new part. We have sometimes used restriction enzymes to do so. All restriction enzymes used were from NEB and digestion were all carried in 1X CutSmart Buffer and incubated 1 hour at 37 Celsius degrees.
 +
</p>
 +
<ul>
 +
  <li>8 µL of Template DNA</li>
 +
  <li>1 µL of 10X Cutsmart Buffer</li>
 +
  <li>1 µL of restriction enzymes (up to three)</li>
 +
  <li>Mix and incubate at 37 Celsius degrees 1 hour</li>
 +
</ul>
 +
 +
<a href="#Biology">Back to Biology Menu</a>
 +
<span id="Gibson"> &nbsp; </span>
 +
<h3>Gison assembly</h3>
 +
<p style=”text-align:justify”>
 +
Gibson assembly was used for all our plasmid construction. It is now a well known technic that uses an exonuclease to degrade 3’->5’ one strand of DNA, permits the homology regions to merge. Than a polymerase will fill the sequence left empty by the exonuclease and a ligase will join both strands together. In our lab, we are using NEBuilder HiFi DNA Assembly cloning kit from NEB. The procedures are specified by the manufacturer, but here if how we do it:
 +
</p>
 +
<ul>
 +
  <li>Design your primers to have at least 20 bp of homology on both sides for the adjacent part on your futur DNA construction. You can use synthetic tags if you want to maximise the success rate.</li>
 +
  <li>Prepare your insert(s) and vector the way you want (digestion, PCR amplification, gBlock synthesis, etc). As long as they have all 20 bp of homology for each other</li>
 +
  <li>Purify all your DNA samples. In our lab, we use Agencourt’s SPRI beads with 1:1 ratio.</li>
 +
  <li>Dose the DNA with a precise method (we use Nanodrop dosage).</li>
 +
  <li>Mix all the DNA fragments at 0,08 pmol of DNA each.</li>
 +
  <li>add water to have 5 µL total.</li>
 +
  <li>Add 5 µL of NEBuilder mix.</li>
 +
  <li>Incubate 1 hour at 55 Celsius degrees.</li>
 +
  <li>Transform all 10 µL in chemically competent cells (heatshock at 42 Celsius degrees for 45 seconds and then recuperate 1 hour at 37 celsius degrees).</li>
 +
  <li>Plate all cells on a LB agar plate containing the right selective antibiotics</li>
 +
</ul>
 +
 +
<a href="#Biology">Back to Biology Menu</a>
  
 
         </div></div> <!--Closing tag for div id="mainContainer" and div id="contentContainer". Opening tag are in the template-->
 
         </div></div> <!--Closing tag for div id="mainContainer" and div id="contentContainer". Opening tag are in the template-->
 
</html>
 
</html>

Revision as of 02:41, 15 September 2015

Experiments & Protocols

Jump to biology section

Projects modules

 

MC96

A thermal experimentation has been the only experimentation done on the MC96 module.

 

Thermal experimentations

The only experimentations done are simulations because no prototype has been built yet.

Simulation

Thermal simulations have been done on the software COMSOL. These simulations have been used to verify the heat transfer of the aluminium mold of the modules, thus helping us improve their design. For the MC96, some simulation has been done on early design, but none on the final design, due to the complexity of simulating heat pipes.

Simulation parameters
Parameters Values
Peltier element cooling power 4X60W
Peltier element heating power 4X250W
Air convective heat transfer coefficient 50W/(m2 ℃)
Isolation conductive heat transfer coefficient 5W/(m ℃)
Aluminium type 6061-t6
Aluminium conductive heat transfer coefficient 167W/(m ℃)
Aluminium specific heat capacity 0.896J/(g ℃)
See Results
Back to MC96
Back to top  

MC1.5

Thermal and magnetisation experimentations have been conduct to validate the design of the MC1.5 module.

 

Thermal experimentation

Simulation

Thermal simulations have been done on the software COMSOL. These simulations have been used to verify the heat transfer of the aluminium mold of the modules, thus helping us improving their design. For the MC1.5, many simulations have been done on different designs. These are the simulation parameters for the latest design.

Simulation parameters
Parameters Values
Peltier element cooling power 60W
Peltier element heating power 250W
Air convective heat transfer coefficient 50W/(m2 ℃)
Isolation conductive heat transfer coefficient 5W/(m ℃)
Aluminium type 6061-t6
Aluminium conductive heat transfer coefficient 167W/(m ℃)
Aluminium specific heat capacity 0.896J/(g ℃)
See Results
Back to MC1.5

Trials protocols

These are the protocol used to test the thermal characteristics of the MC1.5 prototype. These protocols have been tested on a single sub-module of a MC1.5 .


MC1.5 Thermal Experimentations Setup

 

Thermal Experimentations Protocols

 
Maintaining a temperature below room temperature test
Purpose

Determine if the module temperature stability fits the specified of ±1.5℃, when the set temperature is below room temperature. Also, this test determines the voltage versus the set temperature relation.

Material Setup
  1. Connect the power supply (Topward 6303D) to the fan
  2. Power up the power supply and adjust the voltage to 12V
  3. Connect the high current power supply (bk precision 1694 power supply) to the Peltier element (PS vcc to Peltier gnd and PS gnd to Peltier vcc)
  4. Set the thermocouple probe at the bottom of the middle hole of the aluminium mold
  5. Wait for the thermometer measure to stabilize for 20 seconds
Measurement
  1. Set the voltage of the high current power supply to 1V
  2. Wait for thermometer measure to stabilize for at least 20 seconds
  3. Note the thermometer measure and the voltage associated with it
  4. Repeats set 1, 2 and 3 and increased the voltage by 1V each time until the thermometer measure is below the specified lower limit (0℃)
  5. Stop the high current power supply
  6. Stop the fan power supply
See Results
Back to MC1.5 Thermal Experimentations Protocols  
Maintaining a temperature over room temperature test
Purpose

Determine if the module temperature stability fits the specification of ±1.5℃, when the set temperature is over room temperature. Also, this test determines the voltage versus the set temperature relation.

Material Setup
  1. Connect the power supply (Topward 6303D) to the fan
  2. Power up the power supply and adjust the voltage to 12V
  3. Connect the high current power supply (bk precision 1694 power supply) to the Peltier element (PS vcc to Peltier vcc and PS gnd to Peltier gnd)
  4. Set the thermocouple probe at the bottom of the middle hole of the aluminium mold
  5. Wait for the thermometer measure to stabilize for 20 seconds
Measurement
  1. Set the voltage of the high current power supply to 1V
  2. Wait for thermometer measure to stabilize for at least 20 seconds
  3. Note the thermometer measure and the voltage associated with it
  4. Repeats set 1, 2 and 3 and increased the voltage by 1V each time until the thermometer measure is over the specified upper limit (80℃)
  5. Stop the high current power supply
  6. Stop the fan power supply
See Results
Back to MC1.5 Thermal Experimentations Protocols  
Cooling speed test
Purpose

Determine if the module cooling speed fits the specification of 0.5 to 1℃/s. Also, this test determines the optimal voltage to apply to cool the aluminium mold.

Material Setup
  1. Connect the power supply (Topward 6303D) to the fan
  2. Power up the power supply and adjust the voltage to 12V
  3. Connect the high current power supply (bk precision 1694 power supply) to the Peltier element (PS vcc to Peltier vcc and PS gnd to Peltier gnd)
  4. Set the thermocouple probe at the bottom of the middle hole of the aluminium mold
Measurement
  1. Set the voltage of the high current power supply to reach 85℃
  2. Wait for thermometer measure to stabilize for at least 20 seconds
  3. Stop the high current power supply
  4. Invert connection between the Peltier element and the high current power supply
  5. Set the high current power supply to 15.5V (calculated by this method)
  6. Start the chronometer when the thermometer measure reach 80℃
  7. For each 10℃ temperature drop, note the timestamp until 4℃ is reached
  8. Stop the high current power supply
  9. Invert connection between the Peltier element and the high current power supply
  10. Repeats step 1 to 9 for cooling voltage of 15V and 16V
See Results
Back to MC1.5 Thermal Experimentations Protocols  
Theoretical method to determine the optimised cooling voltage
Back to MC1.5 Thermal Experimentations Protocols  
Heating speed test
Purpose

Determine if the module heating speed fits the specified 0.5 to 1℃/s.

Material Setup
  1. Connect the power supply (Topward 6303D) to the fan
  2. Power up the power supply and adjust the voltage to 12V
  3. Connect the high current power supply (bk precision 1694 power supply) to the Peltier element (PS vcc to Peltier gnd and PS gnd to Peltier vcc)
  4. Set the thermocouple probe at the bottom of the middle hole of the aluminium mold
Measurement
  1. Set the voltage of the high current power supply to reach -1℃
  2. Wait for thermometer measure to stabilize for at least 20 second
  3. Stop the high current power supply
  4. Invert connection between the Peltier element and the high current power supply
  5. Set the high current power supply to 24V (Maximal voltage available for the Peltier element)
  6. Start the chronometer when the thermometer measure reach 4℃
  7. For each 10℃ temperature rise, note the timestamp until 80℃ is reached
  8. Stop the high current power supply
See Results
Back to MC1.5 Thermal Experimentations Protocols
Back to Thermal Experimentations
Back to MC1.5
Back to top
 

Magnetisation experimentations

Trials Protocols

These are the protocols used to test the magnetisation characteristics of the MC1.5 prototype. These protocols have been tested on a single sub-module of a MC1.5.

Applying an electromagnetic field on the test tube liquid is one of the key functionality of the MC1.5. This experiment was conduct in order to confirm that the neodymium magnets are powerful enough.

Magnet attraction power test
Purpose

Determine if the neodymium magnets are powerful enough to attract the microscopic magnetic beads on the side of the test tube within 5 minutes.

Material Setup
  1. Agitate the 1.5ml test tube to ensure that the magnetic beads are spreads through the liquid
  2. Place the 1.5ml test tube at the end of the ruler
  3. Place the center of the magnet in the same relative position as in the MC1.5 module (5mm from the bottom of the test tube and 4mm from the side of the test tube)
Measurement
  1. As soon as the magnet is in position, start the chronometer
  2. Stop the chronometer when the liquid has the same transparency as distilled water
  3. Note the timestamp on the chronometer
See Results
Back to MC1.5 Magnetisation Experimentations
Back to MC1.5
Back to top  

TAC

Thermal and turbidity experimentations have been conduct to validate the design of the TAC module.

 

Thermal experimentation

Simulation

Thermal simulations have been done on the software COMSOL. These simulations have been used to verify the heat transfer of the aluminium mold of the modules, thus helping us improving their design. For the TAC, many simulations have been done with different designs. These are the parameters of the simulation for the latest design.

Simulation parameters
Parameters Values
Peltier element cooling power 60W
Peltier element heating power 250W
Air convective heat transfer coefficient 50W/(m2 ℃)
Isolation conductive heat transfer coefficient 5W/(m ℃)
Aluminium type 6061-t6
Aluminium conductive heat transfer coefficient 167W/(m ℃)
Aluminium specific heat capacity 0.896J/(g ℃)
See Results
Back to TAC

Trials protocols

These are the protocol used to test the thermal characteristic of the TAC prototype. These protocols have been tested on a single sub-module of a TAC .


TAC Thermal Experimentations Setup

 

Thermal Experimentations Protocols

 
Maintaining a temperature below room temperature test
Purpose

Determine if the module temperature stability fits the specified of ±1.5℃, when the set temperature is below room temperature. Also, this test determines the voltage versus the set temperature relation.

Material Setup
  1. Connect the power supply (Topward 6303D) to the fan
  2. Power up the power supply and adjust the voltage to 12V
  3. Connect the high current power supply (bk precision 1694 power supply) to the Peltier element (PS vcc to Peltier gnd and PS gnd to Peltier vcc)
  4. Set the thermocouple probe at the bottom of the middle hole of the aluminium mold
  5. Wait for the thermometer measure to stabilize for 20 seconds
Measurement
  1. Set the voltage of the high current power supply to 1V
  2. Wait for thermometer measure to stabilize for at least 20 seconds
  3. Note the thermometer measure and the voltage associated with it
  4. Repeats set 1, 2 and 3 and increased the voltage by 1V each time until the thermometer measure is below the specified lower limit (0℃)
  5. Stop the high current power supply
  6. Stop the fan power supply
See Results
Back to TAC Thermal Experimentations Protocols  
Maintaining a temperature over room temperature test
Purpose

Determine if the module temperature stability fits the specification of ±1.5℃, when the set temperature is over room temperature. Also, this test determines the voltage versus the set temperature relation.

Material Setup
  1. Connect the power supply (Topward 6303D) to the fan
  2. Power up the power supply and adjust the voltage to 12V
  3. Connect the high current power supply (bk precision 1694 power supply) to the Peltier element (PS vcc to Peltier vcc and PS gnd to Peltier gnd)
  4. Set the thermocouple probe at the bottom of the middle hole of the aluminium mold
  5. Wait for the thermometer measure to stabilize for 20 seconds
Measurement
  1. Set the voltage of the high current power supply to 1V
  2. Wait for thermometer measure to stabilize for at least 20 seconds
  3. Note the thermometer measure and the voltage associated with it
  4. Repeats set 1, 2 and 3 and increased the voltage by 1V each time until the thermometer measure is over the specified upper limit (37℃)
  5. Stop the high current power supply
  6. Stop the fan power supply
See Results
Back to TAC Thermal Experimentations Protocols  
Cooling speed test
Purpose

Determine if the module cooling speed fit the specification of 0.3℃/s above room temperature and 0.2℃/s under room temperature. Also, this test determines the optimal voltage to apply to cool the aluminium mold.

Material Setup
  1. Connect the power supply (Topward 6303D) to the fan
  2. Power up the power supply and adjust the voltage to 12V
  3. Connect the high current power supply (bk precision 1694 power supply) to the Peltier element (PS vcc to Peltier vcc and PS gnd to Peltier gnd)
  4. Set the thermocouple probe at the bottom of the middle hole of the aluminium mold
Measurement
  1. Set the voltage of the high current power supply to reach 42℃
  2. Wait for thermometer measure to stabilize for at least 20 seconds
  3. Stop the high current power supply
  4. Invert connection between the Peltier element and the high current power supply
  5. Set the high current power supply to 15.5V (calculated by this method)
  6. Start the chronometer when the thermometer measure reach 37℃
  7. For each 2℃ temperature drop, note the timestamp until 0℃ is reached
  8. Stop the high current power supply
  9. Invert connection between the Peltier element and the high current power supply
  10. Repeats step 1 to 9 for cooling voltage of 15V and 16V
See Results
Back to TAC Thermal Experimentations Protocols  
Heating speed test
Purpose

Determine if the module heating speed fits the specified 0.5 to 1℃/s.

Material Setup
  1. Connect the power supply (Topward 6303D) to the fan
  2. Power up the power supply and adjust the voltage to 12V
  3. Connect the high current power supply (bk precision 1694 power supply) to the Peltier element (PS vcc to Peltier gnd and PS gnd to Peltier vcc)
  4. Set the thermocouple probe at the bottom of the middle hole of the aluminium mold
Measurement
  1. Set the voltage of the high current power supply to reach -5℃
  2. Wait for thermometer measure to stabilize for at least 20 seconds
  3. Stop the high current power supply
  4. Invert connection between the Peltier element and the high current power supply
  5. Set the high current power supply to 24V (Maximal voltage available for the Peltier element)
  6. Start the chronometer when the thermometer measure reach 0℃
  7. For each 5℃ temperature rise, note the timestamp until 37℃ is reached
  8. Stop the high current power supply
See Results
Back to TAC Thermal Experimentations Protocols
Back to TAC Thermal Experimentations
Back to TAC
Back to top
 

Turbidity experimentations

One of the main features of the TAC is the ability to measure the optical density of the liquid inside the test tube. This measure could be used to calculate the population of microorganism in the liquid. This experiment was conducted to calibrate the optical density measurement.

Protocol
Purpose

Calibrate the optical density measurement in the TAC.

Material
  • TAC sub-module
  • Reference test tube filled with liquid with different known optical density
Setup
  1. Power up the TAC module
  2. Start the turbidity function (only amplitude difference is shown on screen)
Measurement
  1. Placed a reference test tube in the TAC's aluminium mold
  2. Note the amplitude difference output
  3. Repeat step 1 and 2 with a different test tube
See Results
Back to TAC
Back to top  

Biology section

 

Biology Experiments

 

Recombineering cassette construction

In order to test our BIOBOT platform (which could ultimately do automated MAGE experiment), a recombineering experiment has to be set-up. While a lot of reliable selectable markers are known, we can’t say the same about counter selectable ones. Many counter selectable markers for recombineering are actually toxins isolated from conjugative plasmids. pVCR94 is a plasmid isolated during the 1994 cholerae outbreak in a Rwanda refugee camp. This conjugative plasmid carries resistance to a lot of antibiotics and its regulation was recently investigated by Carraro et al. 2015.

Like most conjugative plasmids, it possesses many stabilisation and partition systems. One of which is a powerful toxin-anti-toxin system. The toxin was firstly tested for its ability to kill cells compared to other known toxins. To do so, mosT, ccdB, mazF and pVCR94’s toxin: vcrx028 were cloned in pBAD30 to obtain arabinose triggered killswitches. To do so, primers specified in the design sections were used on different template: pSXT, ptac-ccdb-casette, gBlock_FG1, pVCR94. See design section here: Arabinose inducible toxin.

 

Recombineering cassette test

The different killswitches (pBAD30-[mosT;ccdB;mazF;vcrx028]) were tested (see here for the protocol) to investigate if they worked and to know which one was the best. We found that mosT and vcrx028 were the best counter-selectable markers (see here for the results)

Our multi-copy plasmid results shown that mosT and vcrx028 were the best candidates for our system, but we needed to test them in single-integrated-copy for it to be more representative. Therefore, we amplified cassettes containing all the needed elements of the killswitches from pBAD30-[mosT;vcrx028] with primers adding homologies that allow lambda-red mediated recombination in the lacZ truncated gene of Escherichia coli BW25113. After confirmation of the recombinants, they were tested and it shown that both killswitch were still working even in single copy. The main difference is that after 10-12 hours, some mutants that inactivated the integrated killswitch began growing. All the tested clones for mosT shown that behavior, but only 1 of the 3 vcrx028 clones did. See the protocol or See the results

 

Cyclic deletion system test

 

Transfert in Biobrick standardised system

 

Biology Protocols

 

Recombineering cassette test

To test the effect of the toxins on the cells containing the different killswiches:

  • 2 ul of culture was used to inoculate 198 ul of LB-ampicillin-Glucose 5% or LB-ampicillin-arabinose 1%
  • That was done in a 96-wells plate put at 30°C
  • The OD[600] was measured at different intervals during 16-24 hours using a plate reader
Back to Biology Menu  

Medium and reagent use

Medium

The only medium used in this project was LB in broth and agar form. Both comes from BIOBASIC, their catalog numbers are SD7002(S518) and SD7003(S519).

Antibiotics

Here are the complete list of antibiotics used in this project with the working concentration for each:

  • Ampicilin: 100 µg/mL
  • Chloramphenicol: 34 µg/mL
  • Kanmycin: 50 µg/mL

Other Regeants

  • D-Glucose: used at 5% w/v
  • L-Arabinose: used at 1% w/v
  • Molecular Grade Water
  • TFBI:30 mM KOAc, 100 mM RbCl, 10 mM CaCl2, 50 mM MnCl2, 15 % glycerol, pH 5.8 (adjusted with acetic acid), 0.22 µm filtered.
  • TFBII:10 mM MOPS or PIPES, 75 mM CaCl2, 10 mM RbCl, 15 % glycerol, pH 6.5 (adjust with KOH), 0.22 µm filtered.

Strains

  • Escherichia coli K-12 substrain EC100
  • Escherichia coli K-12 substrain BW25113
Back to Biology Menu  

PCR general protocol

The polymerase used for all experiment was Veraseq from Enzymatics™. This polymerase adds 2 kb of DNA per minute. PCR were always done following this fact. All PCR except those specified in the text were made with 50 Celsius degrees as annealing temperature. The recipe for all PCR mix is as follow:

Component Volume for 1 Reaction
Molecular Grade Water 17.5 µL
5X Veraseq Buffer 5 µL
10 mM dNTP 0.5 µL
Forward Primer 0,5 µL
Reverse Primer 0.5 µL
Template DNA (1 ng/µL) 1 µL
Veraseq Polymerase 0.25 µL

The PCR mixes were all done on ice and we followed manufacturer’s recommendation for denaturation and elongation temperature.

Back to Biology Menu  

Chemical transformation

For TFBI and TFBII composition, please refer to other reagents section. Sometimes, chemical transformation can be an advantage compared to electroporation. Basically, the DNA source of the transformation doesn’t need to be pure for a chemical transformation and can be highly concentrated in salt. You can put as much a 10% of the total volume in DNA in each transformation. The cells can be frozen at -80 Celsius degrees and thawed when needed. Here is the total procedure used in our lab to prepare chemically competent cells:

  • Inoculate a single colony into 5 ml LB broth.
  • Incubate 37°C O/N with 200 rpm agitation.
  • Subculture the O/N 1:100 in LB + 6 mM MgSO4 (typically 250 ml).
  • Grow to OD600 = 0,48 (0,4-0,6 is good, should take 2-3h).
  • Centrifuge 6,000 rpm 5 min at 4°C.
  • Gently resuspend pellet in 1/2,5 volume unit ice cold TFBI. (for 250 ml, use 100 ml TFBI ; 50 ml/bottle.
  • . Combine the resuspended cells in one bottle. Keep all steps on ice. Incubate on ice for 5 min.
  • Centrifuge 5,000 rpm 5 min 4°C.
  • Resuspend pellet in 1/25 original volume ice cold TFBII (for 250 ml original, use 10 ml TFBII).
  • Incubate on ice 15-60 min. Put 100 ul per tube and flash freeze tubes with liquid nitrogen. Store at -80°C.
  • When needed, cells can be thawed 15 minutes on ice before using them for transformation
  • Add as much as 10 µL of DNA to a cell aliquot
  • Incubated 45 seconds at 42 Celsius degrees then add 1 mL of cold LB
  • Let cells recuperate 1 hour at 37 degrees before plating them on the right selective plate.
Back to Biology Menu  

Electroporation

To execute this protocol, make sure you have access to an electroporater and be carefull handling the device, as its function is to give electric pulses.

  • Start an overnight pre-culture of your cells to be electroporated at 30 Celsius degrees.
  • Once fully grown (after overnight incubation), inoculated a 4 mL LB broth per needed electroporation by diluting the pre-culture 1:50.
  • Incubate the cells at 30 Celsius egrees with 200 rpm agitation until it has an optical density (OD) of between 0.4 and 0.8 using a 1 cm optic cuvette at 600 nm in a spectrophotometer.
  • Then, transfers your culture in a falcon and incubated on ice for 15 minutes
  • Spin your cells at 7000 x g for 5 minutes.
  • Aspirate the supernatant and wash the pellet with 1 mL of cold sterile water.
  • Transfers the cells in a 1.5 mL microtube and centrifuge at 10 000 x g for 1 minute
  • Get rid of the supernatant and wash again with 1 mL of cold sterile water
  • Centrifuge at 10 000 x g for 1 minute
  • Get rid of the supernatant and wash again with 1 mL of cold sterile water
  • Centrifuge at 10 000 x g for 1 minute
  • Get rid of the supernatant and resuspend your cells in 40 µL of cold sterile water per needed electroporation (which brings them to 1000X concentration)
  • Dispatch 40 µL of cells in microtubes and add 1 µL of DNA 50 ng/µL (if possible).
  • Transfers the cells into 1 mm electroporation cuvettes.
  • Electroporate using 1,8 kV, 200 Ω, 25 µF and target 5.0 ms.
  • Resuspend cells in 1 mL of LB and let them recuperate 1 hour at 30 Celsius degrees (or 37 Celsius degrees).
  • Plate 200 µL of the transformation on a LB agar plate containing the right selection antibiotic.
      • Back to Biology Menu  

      Recombineering

      The recombineering technic ressembles a lot the electroporation one. There is two major adds and changes to the protocol. This comes from the fact that recombineering uses lambda red system. This system is (in our case) carried by a pSIM plasmid. Those plasmid are thermosensible (the origin of replication do not work at 37 degrees Celsius) and the lambda red system is heat induced. Those two fact explains the major changes in the protocol. The recombineering technique was developped by Datsenko et al. (2000) and uses three protein for the lambda phage: bet, gam, exo. exo firstly degrades one of the two strand of a double stranded DNA fragment through its 5’->3’ exonuclease activity. Then, bet will bind the single stranded DNA and insert it in the genome. gam , for instance, will inhibit endogenous nucleases.

      • Start an overnight pre-culture of your cells to be electroporated at 30 Celsius degrees.
      • Once fully grown (after overnight incubation), inoculated a 4 mL LB broth per needed electroporation by diluting the pre-culture 1:50.
      • Incubate the cells at 30 Celsius egrees with 200 rpm agitation until it has an optical density (OD) of between 0.4 and 0.8 using a 1 cm optic cuvette at 600 nm in a spectrophotometer.
      • Transfers the culture into a 42 Celisus degrees agitetive bath and incubate 15 minutes with 180 rpm agitation.
      • Then, transfers your culture in a falcon and incubated on ice for 15 minutes
      • Spin your cells at 7000 x g for 5 minutes.
      • Aspirate the supernatant and wash the pellet with 1 mL of cold sterilewater.
      • Transfers the cells in a 1.5 mL microtube and centrifuge at 10 000 x g for 1 minute
      • Get rid of the supernatant and wash again with 1 mL of cold sterile water
      • Centrifuge at 10 000 x g for 1 minute
      • Get rid of the supernatant and wash again with 1 mL of cold sterile water
      • Centrifuge at 10 000 x g for 1 minute
      • Get rid of the supernatant and resuspend your cells in 40 µL of cold sterile water per needed electroporation (which brings them to 1000X concentration)
      • Dispatch 40 µL of cells in microtubes and add 1 µL of DNA 50 ng/µL (if possible).
      • Transfers the cells into 1 mm electroporation cuvettes.
      • Electroporate using 1,8 kV, 200 Ω, 25 µF and target 5.0 ms.
      • Resuspend cells in 1 mL of LB and let them recuperate 1 hour at 30 Celsius degrees (or 37 Celsius degrees if you want to loose the pSIM plasmid).
      • Plate all of the transformation on a LB agar plate containing the right selection antibiotic (you can spin the cells and resuspend them in 100 µL to concentrate them).
          • Back to Biology Menu  

          DNA miniprep

          The DNA minipreps were all done using the EZ-10 Spin Colomn Miniprep Kit™. Those are available commercially and distributed by Biobasic™. We have strictly followed manufacturer’s recomendation and obtained 50 µL of 50 ng/µL on average.

          Back to Biology Menu  

          DNA digestion with restriction enzymes

          It may be necessary in some cases to digest the vector in order to construct a new part. We have sometimes used restriction enzymes to do so. All restriction enzymes used were from NEB and digestion were all carried in 1X CutSmart Buffer and incubated 1 hour at 37 Celsius degrees.

          • 8 µL of Template DNA
          • 1 µL of 10X Cutsmart Buffer
          • 1 µL of restriction enzymes (up to three)
          • Mix and incubate at 37 Celsius degrees 1 hour
          Back to Biology Menu  

          Gison assembly

          Gibson assembly was used for all our plasmid construction. It is now a well known technic that uses an exonuclease to degrade 3’->5’ one strand of DNA, permits the homology regions to merge. Than a polymerase will fill the sequence left empty by the exonuclease and a ligase will join both strands together. In our lab, we are using NEBuilder HiFi DNA Assembly cloning kit from NEB. The procedures are specified by the manufacturer, but here if how we do it:

          • Design your primers to have at least 20 bp of homology on both sides for the adjacent part on your futur DNA construction. You can use synthetic tags if you want to maximise the success rate.
          • Prepare your insert(s) and vector the way you want (digestion, PCR amplification, gBlock synthesis, etc). As long as they have all 20 bp of homology for each other
          • Purify all your DNA samples. In our lab, we use Agencourt’s SPRI beads with 1:1 ratio.
          • Dose the DNA with a precise method (we use Nanodrop dosage).
          • Mix all the DNA fragments at 0,08 pmol of DNA each.
          • add water to have 5 µL total.
          • Add 5 µL of NEBuilder mix.
          • Incubate 1 hour at 55 Celsius degrees.
          • Transform all 10 µL in chemically competent cells (heatshock at 42 Celsius degrees for 45 seconds and then recuperate 1 hour at 37 celsius degrees).
          • Plate all cells on a LB agar plate containing the right selective antibiotics
          Back to Biology Menu